Urine biomarkers for detecting graft rejection

ABSTRACT

Provided herein are compositions, systems, kits, and methods for detecting rejection, or an elevated risk of rejection, of an organ or tissue graft (e.g., kidney graft) in a subject by detecting one, or a panel of, RNA markers in a urine sample from the subject. In certain embodiments, kidney graft injury is detected by measuring one or more of the RNA markers disclosed herein. In some embodiments, the one or more markers are employed to distinguish between acute graft rejection and BK virus nephropathy.

The present application is a Continuation of U.S. patent applicationSer. No. 16/110,964, filed Aug. 23, 2018, now allowed, which is aContinuation-in-part of U.S. patent application Ser. No. 16/004,055filed Jun. 8, 2018, now abandoned, which claims priority to U.S.Provisional application 62/516,907 filed Jun. 8, 2017, both of which areherein incorporated by reference in their entireties as if fully setforth herein.

STATEMENT REGARDING GOVERNMENT FUNDING

This invention was made with government support under A1084150 andA1063594 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

Provided herein are compositions, systems, kits, and methods fordetecting rejection, or an elevated risk of rejection, of an organ ortissue graft (e.g., kidney graft) in a subject by detecting one, or apanel of, RNA markers in a urine sample from the subject. In certainembodiments, kidney graft injury is detected by measuring one or more ofthe RNA markers disclosed herein. In some embodiments, the one or moremarkers are employed to distinguish between acute graft rejection and BKvirus nephropathy.

BACKGROUND

Kidney disease and failure are common medical problems associated withmany illnesses. One in three Americans is considered to be at risk forkidney disease. The predictors include age above 60, diabetes, highblood pressure, and family history of kidney disease. Kidney disease canbe chronic (ongoing over years with slowly declining kidney function) oracute (sharp decline in kidney function, either nearly immediate orwithin months). When kidney function declines to a level of reducedeffectiveness, this leaves the patient with only 10-15% of kidneyfunction remaining, giving a diagnosis of kidney failure to the patient.Common treatments for kidney failure include dialysis and kidneytransplants. In 2014, about 17,000 kidney transplants occurred in theUS. Of these, 11,570 kidneys came from deceased donors and 5,537 camefrom living donors. Typically, within the first year of a kidneytransplant, there is a 7% failure rate, and within 10 years of atransplant, there is a 45% failure rate. In addition, kidney supply isnot fulfilling kidney demand leading to patient death due to notreceiving a transplant in time. In 2014, nearly 5,000 patients diedwhile waiting for a transplant, on a waiting list, and more than 3,600became too sick to receive a transplant.

Transplanted kidneys fail for various reasons, including kidneyrejection. Rejection can be acute, very soon after surgery, and itoccurs in about 15% of cases. There is also chronic rejection, whichtakes place over longer periods of time, generally taking years. Mostpatients who experience kidney rejection do not lose the transplantedkidney but instead go on to other treatments to manage the immunesystem's rejection. Accurate, early detection of the rejection can leadto better early intervention and treatment of patients. In addition,successful transplants are less expensive than dialysis, in most cases,and lead patients to live longer, healthier lives. Each year one in fivetransplants is performed into a patient who had previously received atransplant and is either suffering from kidney failure or kidneytransplant rejection. For patients that experience transplant rejectionand are unable to get another organ, they receive dialysis at a cost of$70,000 per year in the US.

Effective monitoring of kidney transplant patients for rejection is acontinuing problem. As of 2016, the most effective methods of testingfor transplanted kidney failure are generally considered to be through acombination of monitoring non-specific byproducts of kidney rejection,such as creatinine levels. Physicians further make a clinical assessmentof a variety of patient symptoms commonly associated with kidneytransplant rejection, including flu-like symptoms, chills, body aches,nausea, cough, shortness of breath, and a general sense of unease, 10followed by or in concert with a biopsy of the kidney. Kidney biopsiesare not considered a viable option for continuous, regular monitoring,and efforts to determine kidney rejection through less invasive means,including blood and urine testing, are continuously increasing. Althoughthere have been various blood and urine tests developed during the lastseveral years, as of March 2016, there does not seem to be a test thathas been adopted for widespread clinical use. This is in part becausekidney rejection is believed to be a heterologous process with manycauses, depending on compatibility of the donor and recipient, and otherreasons as well. As a result, some in the medical community do notbelieve a uniform, accurate test is possible. Monitoring of thedifferent byproducts of kidney disease, such as serum creatinineantibodies in the blood have been attempted through blood and urinetests, but none appears to have been generally adopted as a new goldstandard by the medical community. None in use so far has beenestablished to detect kidney rejection across all types of transplantpatients.

A combination of creatinine monitoring and biopsy is a frequently usedapproach in medical environments. Acute cellular rejection is asignificant type of kidney organ rejection that requires furthertreatment to the patient to compensate for rejection, or, at times,removal of the rejected kidney.

SUMMARY

Provided herein are compositions, systems, kits, and methods fordetecting rejection, or an elevated risk of rejection, of an organ ortissue graft (e.g., kidney graft) in a subject by detecting one, or apanel, of RNA markers in a urine sample from the subject. In certainembodiments, kidney graft injury is detected by measuring one or more ofthe RNA markers disclosed herein. In some embodiments, the one or moremarkers are employed to distinguish between acute graft rejection and BKvirus nephropathy.

In some embodiments, provided herein are methods for detectingrejection, or an elevated risk of rejection, or an organ or tissue graftin a subject comprising: detecting an upregulated level of at least onefirst RNA marker (e.g., 1 . . . 10 . . . 25 . . . 35) in a sample from asubject and/or a downregulated level of at least one second RNA marker(e.g., 1 . . . 10 . . . 25 . . . 35) in said sample from said subject,and thereby detecting acute rejection and/or elevated risk of rejectionof said organ or tissue graft or detecting kidney graft injury, ordistinguishing between acute graft rejection and BK virus nephropathy,wherein said sample comprises urine (or other sample type, such as serumor plasma) from said subject, wherein said subject has a tissue or organgraft, wherein said at least one first RNA marker that is detected asbeing upregulated is selected from the group consisting of: CXCL10,CXCL9, CXCL11, PRF1, CCL5, CX3CL1, IDO1, GZMA, LAG3, CD3D, CD27, KLRK1,GZMK, MMP7, CD8A, GZMB, ETS1, CXCR6, CCL2, IL2RB, CTLA4, MSR1, FYN,ITGA6, CD3E, LCK, CD2, IRF8, CD3G, CD247, GZMH, ITK, CD38, CDK1, ZAP70,LIF, CD7, CD40, CD6, IL21R, SIGIRR, CDH1, CD81, CD74, STAT1, AXL, TBX21,COG7, TCRA, C1QB, NKG7, THBS1, C1QA, EPCAM, DPP4, HLA-DPA1, TIGIT,HLA-DPB1, CXCL13, C1R, CD5, GPR171, EFEMP1, HLA-DMA, LY9, CCL8, CD96,HAVCR2, NFATC2, LAMC2, IFNG, STAT4, APOE, C4B, CTSL, SOCS1, PVR, CCR5,CD86, TNFSF12, SIGLEC1, ST6GAL1, C3, KLRC2, CTSW, CARD11, GNLY, PSMB10,ICOS, TAP1, TNFRSF11B, ITGA1, IRF4, CD4, CD84, CSF1, SH2D1A, CD8B, CD47,ABCB1, SLAMF1, HLA-DMB, IFI35, A2M, HLA-DRA, C1QBP, SPP1, VCAM1, NELL2,FLT3LG, ITGA4, TNFRSF10B, RUNX3, SERPING1, KLRC1, NLRC5, HPRT1, IKBKE,TNF, ADA PDCD1, C1S, CCR2, TAB1, IL12RB1, IL2RA, PSEN2, LRP1, NOL7, SPN,TCF7, MFGE8, MST1R, IL10, LRRN3, PSMB9, PPIA, AGK, EDC3, CD80, PSMB8,CD276, BIRC5, TNFRSF9, TP53, ZC3H14, HLA-DRB3, ICOSLG, CD200, PIN1,EGR2, ITGAE, TRAF2, ABL1, ALCAM, IRF5, SF3A3, HRAS, NOD1, PDCD1LG2,IL16, LILRB1, and IRF3, and wherein said at least one second RNA markerthat is detected as being downregulated is selected from the groupconsisting of: ANXA1, BASP1, BCL6, BLK, CAMP, CCL17, CCL22, CCR3,CEACAM1, CEACAM6, CFD, CR2, CSF2RB, CSF3R, CXCL1, CXCL6, CXCR1, CXCR2,FUT7, IFNL1, IL18RAP, IL19, ILIA, IL1RAP, IL1RN, IL8, ITGAX, LCN2,LILRB3, LYN, MAPK3, MEFV, MYD88, OSM, PBEF1, POLR2A, PRKCD, PTGS2,S100A12, S100A7, S100A8, SH2B2, SH2D1B, SPINK5, TFE3, TLR1, TNFRSF10C,TNFRSF11A, TNFRSF1A, TNFSF14, ADAMS, ARG2, BCL10, CASP3, CD46, CFI,DMBT1, DOCKS, DUSP4, IFI27, IKBKB, IL23A, ISG15, ITCH, LGALS3, MAPK8,MUC1, NOS2A, SERPINA3, SERPINB2, SYT17, and TREM1.

In certain embodiments, the subject, at the time the sample wasobtained, is taking a first immunosuppressant. In particularembodiments, the method further comprises the step of performing atleast one of the following: i) treating said subject with a secondimmunosuppressant that is different from said first immunosuppressant;ii) generating and/or transmitting a report that indicates said at leastone first RNA maker is upregulated and/or said at least one second RNAmarker is downregulated in said sample, and that said subject is in needof an immunosuppressant different from said first immunosuppressantand/or is in need of a having said tissue and/or organ graft removed(and/or in need of having said tissue or graft replaced with a differenttissue or graft); iii) generating and/or transmitting a report thatindicates said at least one first RNA maker is upregulated and/or saidat least one second RNA marker is downregulated in said sample, and thatsaid subject has acute rejection and/or elevated risk of rejection ofsaid organ or tissue graft; and iv) characterizing said subject ashaving acute rejection and/or an elevated risk of rejection of saidorgan or tissue graft, based on finding that said at least one first RNAmaker is upregulated and/or said at least one second RNA marker isdownregulated in said sample.

In particular embodiments, the organ or tissue graft comprises a kidneyor kidney tissue graft. In further embodiments, the subject at the timeof collection of said sample was being treated with, and/or wherein saidsample comprises, an immunosuppressant protein that comprises anextracellular domain of CTLA-4. In further embodiments, theimmunosuppressant protein further comprises an Fc region. In otherembodiments, the immunosuppressant protein comprises Belatacept orAbatacept. In other embodiments, the detecting at least one of saidfirst and/or said at least one second RNA marker comprises detecting atleast two (e.g., 2 . . . 5 . . . 10 . . . 15 . . . 20 . . . 25) of saidfirst and/or second RNA markers. In certain embodiments, the methodsfurther comprise detecting an additional marker selected fromupregulated TGFB1 and/or upregulated HLA.A. In other embodiments, themethods further comprise detecting an additional marker selected fromupregulated CXCL9 and/or upregulated CXCL10. In other embodiments, themethods further comprise detecting all of the following: i) upregulatedCXCL11, TGFB1, LIF, CCL5, TNF, HLA.A, and ETS1, and ii) downregulatedIL19, CCL17, and IFIT1.

In certain embodiments, the provided herein are kits, compositions, andsystems comprising: a) a sample from a subject having a tissue or organgraft, wherein said sample comprises urine (or other sample type, suchas serum, plasma, whole blood, etc.) from said subject; and b) reagents(e.g., sequencing reagents, nucleic acid probes, RT enzymes,polymerases, detectable labels, expression panels, NANOSTRINGS reagents,etc.) configured for detecting the level of at least one RNA markerselected from the group consisting of: CXCL10, CXCL9, CXCL11, PRF1,CCL5, CX3CL1, IDO1, GZMA, LAG3, CD3D, CD27, KLRK1, GZMK, MMP7, CD8A,GZMB, ETS1, CXCR6, CCL2, IL2RB, CTLA4, MSR1, FYN, ITGA6, CD3E, LCK, CD2,IRF8, CD3G, CD247, GZMH, ITK, CD38, CDK1, ZAP70, LIF, CD7, CD40, CD6,IL21R, SIGIRR, CDH1, CD81, CD74, STAT1, AXL, TBX21, COG7, TCRA, C1QB,NKG7, THBS1, C1QA, EPCAM, DPP4, HLA-DPA1, TIGIT, HLA-DPB1, CXCL13, C1R,CD5, GPR171, EFEMP1, HLA-DMA, LY9, CCL5, CD96, HAVCR2, NFATC2, LAMC2,IFNG, STAT4, APOE, C4B, CTSL, SOCS1, PVR, CCR5, CD86, TNFSF12, SIGLEC1,ST6GAL1, C3, KLRC2, CTSW, CARD11, GNLY, PSMB10, ICOS, TAP1, TNFRSF11B,ITGA1, IRF4, CD4, CD84, CSF1, SH2D1A, CD8B, CD47, ABCB1, SLAMF1,HLA-DMB, IFI35, A2M, HLA-DRA, C1QBP, SPP1, VCAM1, NELL2, FLT3LG, ITGA4,TNFRSF10B, RUNX3, SERPING1, KLRC1, NLRC5, HPRT1, IKBKE, TNF, ADA PDCD1,C1S, CCR2, TAB1, IL12RB1, IL2RA, PSEN2, LRP1, NOL7, SPN, TCF7, MFGE8,MST1R, IL10, LRRN3, PSMB9, PPIA, AGK, EDC3, CD80, PSMB8, CD276, BIRC5,TNFRSF9, TP53, ZC3H14, HLA-DRB3, ICOSLG, CD200, PIN1, EGR2, ITGAE,TRAF2, ABL1, ALCAM, IRF5, SF3A3, HRAS, NOD1, PDCD1LG2, IRF3, ANXA1,BASP1, BCL6, BLK, CAMP, CCL17, CCL22, CCR3, CEACAM1, CEACAM6, CFD, CR2,CSF2RB, CSF3R, CXCL1, CXCL6, CXCR1, CXCR2, FUT7, IFNL1, IL18RAP, IL19,IL1A, IL1RAP, IL1RN, IL8, ITGAX, LCN2, LILRB3, LYN, MAPK3, MEFV, MYD88,OSM, PBEF1, POLR2A, PRKCD, PTGS2, S100A12, S100A7, S100A8, SH2B2,SH2D1B, SPINK5, TFE3, TLR1, TNFRSF10C, TNFRSF11A, TNFRSF1A, TNFSF14,ADAMS, ARG2, BCL10, CASP3, CD46, CFI, DMBT1, DOCKS, DUSP4, IFI27, IKBKB,IL23A, ISG15, ITCH, LGALS3, MAPK8, MUC1, NOS2A, SERPINA3, SERPINB2,SYT17, IL16, LILRB1, and TREM1.

In some embodiments, provided herein are kits, compositions, and systemscomprising: a) a sample from a subject having a tissue or organ graft,wherein said sample comprises urine (or other sample type, such asserum, plasma, whole blood, etc.) from said subject; and b) reagents(e.g., sequencing reagents, nucleic acid probes, RT enzymes,polymerases, detectable labels, expression panels, NANOSTRINGS reagents,etc.) configured for detecting the level of at least one RNA markerselected from the group consisting of: CXCL11, TGFB1, LIF, CCL5, TNF,HLA.A, and ETS1, IL19, CCL17, and IFIT1.

In some embodiments, said urine sample is from a subject having a kidneytissue or organ graft. In certain embodiments, the subject at the timeof collection of said sample was being treated with, and/or wherein saidsample comprises, an immunosuppressant protein that comprises anextracellular domain of CTLA-4. In further embodiments, theimmunosuppressant protein further comprises an Fc region. In additionalembodiments, the immunosuppressant protein comprises Belatacept orAbatacept.

In some embodiments, provided herein are methods of detecting rejection,or an elevated risk of rejection, of an organ or tissue graft, ordetecting kidney graft injury, or distinguishing between acute graftrejection and BK virus nephropathy, in a subject comprising: detectingan upregulated level of at least one first RNA marker (e.g., 1 . . . 10. . . 25 . . . 35) in a sample from a subject and/or a downregulatedlevel of at least one second RNA marker (e.g., 1 . . . 10 . . . 25 . . .35) in said sample from said subject, and thereby detecting acuterejection and/or elevated risk of rejection of said organ or tissuegraft, wherein said sample comprises urine (or other sample, such asblood, plasma, whole blood, or tissue biopsy) from said subject, whereinsaid subject has a tissue or organ graft, wherein said at least onefirst RNA marker that is detected as being upregulated is selected fromthe group consisting of: TNFRSF11B, ETS1, CXCL10, CDK1, CXCL9, SIGIRR,CD274, CXCL11, ITGA6, and SF3A3, and wherein said at least one secondRNA marker that is detected as being downregulated, are selected fromthe group consisting of: TNFSF4, ATG16L1, MERTK, DHX16, CXCL14, IL19,CCL17, ATF1, CFD, IFIT1, CD163, ELK1, DPP4, and LAMP2. In certainembodiments, the methods further comprise detecting an additional markerselected from upregulated CXCL9 and/or upregulated CXCL10.

In particular embodiments, the subject was taking a firstimmunosuppressant at the time said sample was obtained. In furtherembodiments, the methods further comprise the step of performing atleast one of the following: i) treating said subject with a secondimmunosuppressant that is different from said first immunosuppressant;ii) generating and/or transmitting a report that indicates said at leastone first RNA maker is upregulated and/or said at least one second RNAmarker is downregulated in said sample, and/or that said subject is inneed of an immunosuppressant different from said firstimmunosuppressant, and/or is in need of a having said tissue and/ororgan graft removed or replaced; iii) generating and/or transmitting areport that indicates said at least one first RNA maker is upregulatedand/or said at least one second RNA marker is downregulated in saidsample, and/or that said subject has acute rejection and/or elevatedrisk of rejection of said organ or tissue graft; and iv) characterizingsaid subject as having acute rejection and/or an elevated risk ofrejection of said organ or tissue graft, based on finding that said atleast one first RNA maker is upregulated and/or said at least one secondRNA marker is downregulated in said sample.

In certain embodiments, said organ or tissue graft comprises a kidney orkidney tissue graft. In further embodiments, the subject has beentreated with an anti-CD52 monoclonal antibody or antigen bindingfragment thereof. In additional embodiments, the anti-CD52 monoclonalantibody comprises Alemtuzumab/CAMPATH. In some embodiments, the subjectat the time of collection of said sample was being treated with, and/orwherein said sample comprises, an immunosuppressant comprisingtacrolimus (aka FK506). In some embodiments, the at least one of saidfirst and/or second RNA marker comprises detecting at least two (e.g., 2. . . 5 . . . 10 . . . 15 . . . 25 . . . 35) of said first and/or secondRNA markers. In other embodiments, the detecting at least one of saidfirst and/or second RNA marker comprises detecting at least four of saidfirst and/or second RNA markers.

In some embodiments, provided herein are compositions, kits, or systemscomprising: a) a sample from a subject having a tissue or organ graft,wherein said sample comprises urine from said subject; and b) reagentconfigured for detecting the level of at least one RNA marker selectedfrom the group consisting of: CCL17, CD163, CD274, CDK1, CFD, CXCL10,CXCL11, CXCL14, CXCL9, DHX16, DPP4, ELK1, ETS1, IFIT1, IL19, ITGA6,LAMP2, MERTK, SF3A3, SIGIRR, TNFRSF11B, and TNFSF4.

In certain embodiments, the urine sample is from a subject having akidney tissue or organ graft. In further embodiments, the subject hasbeen treated with an anti-CD52 monoclonal antibody or antigen bindingfragment thereof for immunosuppression of said tissue or graft. In otherembodiments, the anti-CD52 monoclonal antibody comprisesAlemtuzumab/CAMPATH. In other embodiments, the subject at the time ofcollection of said sample was being treated with, and/or wherein saidsample comprises, an immunosuppressant comprising tacrolimus (akaFK506).

In some embodiments, provided herein are methods of detecting rejectionor BK virus infection (e.g., distinguishing between acute rejection andBK virus infection), or an elevated risk of rejection, of an organ ortissue graft in a subject comprising: detecting an upregulated level ofat least one first RNA marker in a sample from a subject and/or adownregulated level of at least one second RNA marker in said samplefrom said subject, and thereby detecting acute rejection and/or elevatedrisk of rejection of said organ or tissue graft, wherein said samplecomprises urine from said subject, wherein said subject has a tissue ororgan graft, wherein said at least one first RNA marker that is detectedas being upregulated and is selected from the group consisting of:SIGLEC1, CD163, PSEN1, DPP4, CEACAM8, PYCARD, MTMR14, ICOSLG, NOL7, C2,HLA.DRB3, CD68, ST6GAL1, SLAMF1, MERTK, PRAME, ELK1, STAT4, CD99, CD86,LAMP2, ITGB1, IL21R, IL6ST, TNF, GPATCH3, GPI, ATF1, PIN1, and YTHDF2,and wherein said at least one second RNA marker that is detected asbeing downregulated, is selected from the group consisting of: HMGB1,MAPK9, SPA17, TNFRSF11B, GTF3C1, BST2, PRPF38A, MAF, BATF, EGFR, CD274,CCL17, SERPINA3, GBP1, and PBEF1. In some embodiments, the methodsfurther comprise detecting an additional marker selected from the groupconsisting of: C1QBP, CD6, CXCL6, LIF, and AXL. In other embodiments,the methods further comprise detecting an additional marker selectedfrom the group consisting of: CREBBP, PSMB7, NFATC1, TGFB1, and FCER1G.In other embodiments, the detecting comprise detecting all of thefollowing: SIGLEC1, CD163, PSEN1, DPP4, CEACAM8, PYCARD, MTMR14, ICOSLG,NOL7, C2, HLA.DRB3, CD68, ST6GAL1, SLAMF1, MERTK, PRAME, ELK1, STAT4,CD99, CD86, LAMP2, ITGB1, IL21R, IL6ST, TNF, GPATCH3, GPI, ATF1, PIN1,YTHDF2, HMGB1, MAPK9, SPA17, TNFRSF11B, GTF3C1, BST2, PRPF38A, MAF,BATF, EGFR, CD274, CCL17, SERPINA3, GBP1, PBEF1, C1QBP, CD6, CXCL6, LIF,AXL, CREBBP, PSMB7, NFATC1, TGFB1, and FCER1G.

In certain embodiments, the BK virus infection is detected (anddistinguished from acute rejection). In other embodiments, acuterejection is detected (e.g., and distinguished from BK virus infection).In some embodiments, the organ or tissue graft comprises a kidney orkidney tissue graft. In additional embodiments, the subject has beentreated with an anti-CD52 monoclonal antibody or antigen bindingfragment thereof. In some embodiments, the anti-CD52 monoclonal antibodycomprises Alemtuzumab/CAMPATH. In certain embodiments, said subject atthe time of collection of said sample was being treated with, and/orwherein said sample comprises, an immunosuppressant comprisingtacrolimus (aka FK506).

In some embodiments, provided herein are kits, compositions, and systemscomprising: a) a sample from a subject having a tissue or organ graft,wherein said sample comprises urine from said subject; and b) reagentconfigured for detecting the level of at least one RNA marker selectedfrom the group consisting of: ATF1, BATF, BST2, C2, CCL17, CD163, CD274,CD68, CD86, CD99, CEACAM8, DPP4, EGFR, ELK1, GBP1, GPATCH3, GPI, GTF3C1,HLA.DRB3, HMGB1, ICOSLG, IL21R, IL6ST, ITGB1, LAMP2, MAF, MAPK9, MERTK,MTMR14, NOL7, PBEF1, PIN1, PRAME, PRPF38A, PSEN1, PYCARD, SERPINA3,SIGLEC1, SLAMF1, SPA17, ST6GAL1, STAT4, TNF, TNFRSF11B, and YTHDF2.

In some embodiments, provided herein are kits, compositions, and systemscomprising: a) a sample from a subject having a tissue or organ graft,wherein said sample comprises urine from said subject; and b) reagentsconfigured for detecting the level of at least one (e.g., 1 . . . 10 . .. 25 . . . 35 . . . 45 . . . or more) RNA marker selected from the groupconsisting of: SIGLEC1, CD163, PSEN1, DPP4, CEACAM8, PYCARD, MTMR14,ICOSLG, NOL7, C2, HLA.DRB3, CD68, ST6GAL1, SLAMF1, MERTK, PRAME, ELK1,STAT4, CD99, CD86, LAMP2, ITGB1, IL21R, IL6ST, TNF, GPATCH3, GPI, ATF1,PIN1, YTHDF2, HMGB1, MAPK9, SPA17, TNFRSF11B, GTF3C1, BST2, PRPF38A,MAF, BATF, EGFR, CD274, CCL17, SERPINA3, GBP1, PBEF1, C1QBP, CD6, CXCL6,LIF, AXL, CREBBP, PSMB7, NFATC1, TGFB1, and FCER1G.

In certain embodiments, the urine sample is from a subject having akidney tissue or organ graft. In further embodiments, the subject hasbeen treated with an anti-CD52 monoclonal antibody or antigen bindingfragment thereof. In additional embodiments, the anti-CD52 monoclonalantibody comprises Alemtuzumab/CAMPATH. In some embodiments, the subjectat the time of collection of said sample was being treated with, and/orwherein said sample comprises, an immunosuppressant comprisingtacrolimus (aka FK506).

In some embodiments, provided herein are methods of detecting rejection,or an elevated risk of rejection, or an organ or tissue graft in asubject comprising: detecting an upregulated level of at least one firstRNA marker (e.g., mRNA sequence or portion thereof) in a sample from asubject and/or a downregulated level of at least one second RNA marker(e.g., mRNA sequence or portion thereof) in the sample from the subject,and thereby detecting acute rejection and/or elevated risk of rejectionof the organ or tissue graft, wherein the sample comprises urine fromthe subject, wherein the subject has a tissue or organ graft, whereinthe first RNA marker (e.g., mRNA sequence or portion thereof) that isdetected as being upregulated is selected from the group consisting of:CXCL10, CXCL9, CXCL11, PRF1, CCL5, CX3CL1, IDO1, GZMA, LAG3, CD3D, CD27,KLRK1, GZMK, MMP7, CD8A, GZMB, ETS1, CXCR6, CCL2, IL2RB, CTLA4, MSR1,FYN, ITGA6, CD3E, LCK, CD2, IRF8, CD3G, CD247, GZMH, ITK, CD38, CDK1,ZAP70, LIF, CD7, CD40, CD6, IL21R, SIGIRR, CDH1, CD81, CD74, STAT1, AXL,TBX21, COG7, TCRA, C1QB, NKG7, THBS1, C1QA, EPCAM, DPP4, HLA-DPA1,TIGIT, HLA-DPB1, CXCL13, C1R, CD5, GPR171, EFEMP1, HLA-DMA, LY9, CCL8,CD96, HAVCR2, NFATC2, LAMC2, IFNG, STAT4, APOE, C4B, CTSL, SOCS1, PVR,CCR5, CD86, TNFSF12, SIGLEC1, ST6GAL1, C3, KLRC2, CTSW, CARD11, GNLY,PSMB10, ICOS, TAP1, TNFRSF11B, ITGA1, IRF4, CD4, CD84, CSF1, SH2D1A,CD8B, CD47, ABCB1, SLAMF1, HLA-DMB, IFI35, A2M, HLA-DRA, C1QBP, SPP1,VCAM1, NELL2, FLT3LG, ITGA4, TNFRSF10B, RUNX3, SERPING1, KLRC1, NLRC5,HPRT1, IKBKE, TNF, ADA, PDCD1, C1S, CCR2, TAB1, IL12RB1, IL2RA, PSEN2,LRP1, NOL7, SPN, TCF7, MFGE8, MST1R, IL10, LRRN3, PSMB9, PPIA, AGK,EDC3, CD80, PSMB8, CD276, BIRC5, TNFRSF9, TP53, ZC3H14, HLA-DRB3,ICOSLG, CD200, PIN1, EGR2, ITGAE, TRAF2, ABL1, ALCAM, IRF5, SF3A3, HRAS,NOD1, PDCD1LG2, and IRF3; and wherein the second RNA marker (e.g., mRNAsequence or portion thereof) that is detected as being downregulated isselected from the group consisting of: ANXA1, BASP1, BCL6, BLK, CAMP,CCL17, CCL22, CCR3, CEACAM1, CEACAM6, CFD, CR2, CSF2RB, CSF3R, CXCL1,CXCL6, CXCR1, CXCR2, FUT7, IFNL1, IL18RAP, IL19, IL1A, IL1RAP, IL1RN,IL8, ITGAX, LCN2, LILRB3, LYN, MAPK3, MEFV, MYD88, OSM, PBEF1, POLR2A,PRKCD, PTGS2, S100A12, S100A7, S100A8, SH2B2, SH2D1B, SPINK5, TFE3,TLR1, TNFRSF10C, TNFRSF11A, TNFRSF1A, TNFSF14, and TREM1.

In particular embodiments, provided herein are methods of detectingrejection, or an elevated risk of rejection, or an organ or tissuegraft, or detecting kidney graft injury, or distinguishing between acutegraft rejection and BK virus nephropathy, in a subject comprising:detecting an upregulated level of at least one RNA marker in a samplefrom a subject, and thereby detecting acute rejection and/or elevatedrisk of rejection of the organ or tissue graft, wherein the samplecomprises urine from the subject, wherein the subject has a tissue ororgan graft, wherein the RNA marker that is detected as beingupregulated is selected from the group consisting of: CXCL9, CXCL10,CXCL11, LAG3, CD38, CD3D, IDOL CCL5, PRF1, KLRK1, TCRA, CTLA4, CD8A,STAT4, and CD27.

In certain embodiments, provided herein are methods of detectingrejection, or an elevated risk of rejection, or an organ or tissue graftin a subject comprising: detecting an upregulated level of at least oneRNA marker in a sample from a subject, and thereby detecting acuterejection and/or elevated risk of rejection of the organ or tissuegraft, wherein the sample comprises urine from the subject, wherein thesubject has a tissue or organ graft, wherein the RNA marker that isdetected as being upregulated is selected from the group consisting of:C1QA, C1QB, C1R, CD3E, CTSL, CX3CL1, GSMA, HAVCR2, IF135, MSR1, SIGLEC1,and ENG.

In some embodiments, the organ or tissue graft comprises a kidney orkidney tissue graft or a liver or liver tissue. In particularembodiments, the detecting at least one of the first and/or second RNAmarker comprises detecting at least two, three, four, five, six, seven,. . . twenty . . . thirty . . . forty . . . or seventy-five of the firstand/or second RNA markers. In certain embodiments, a Nanostrings assayis employed to detect the first and/or second RNA markers.

In particular embodiments, provided herein are kits, systems, and/orcompositions comprising: a) a sample from a subject having a tissue ororgan graft, wherein the sample comprises urine from the subject; and b)reagent configured for detecting the level of at least one RNA marker(e.g., mRNA sequence or portion thereof) selected from the groupconsisting of: CXCL10, CXCL9, CXCL11, PRF1, CCL5, CX3CL1, IDO1, GZMA,LAG3, CD3D, CD27, KLRK1, GZMK, MMP7, CD8A, GZMB, ETS1, CXCR6, CCL2,IL2RB, CTLA4, MSR1, FYN, ITGA6, CD3E, LCK, CD2, IRF8, CD3G, CD247, GZMH,ITK, CD38, CDK1, ZAP70, LIF, CD7, CD40, CD6, IL21R, SIGIRR, CDH1, CD81,CD74, STAT1, AXL, TBX21, COG7, TCRA, C1QB, NKG7, THBS1, C1QA, EPCAM,DPP4, HLA-DPA1, TIGIT, HLA-DPB1, CXCL13, C1R, CD5, GPR171, EFEMP1,HLA-DMA, LY9, CCL8, CD96, HAVCR2, NFATC2, LAMC2, IFNG, STAT4, APOE, C4B,CTSL, SOCS1, PVR, CCR5, CD86, TNFSF12, SIGLEC1, ST6GAL1, C3, KLRC2,CTSW, CARD11, GNLY, PSMB10, ICOS, TAP1, TNFRSF11B, ITGA1, IRF4, CD4,CD84, CSF1, SH2D1A, CD8B, CD47, ABCB1, SLAMF1, HLA-DMB, IFI35, A2M,HLA-DRA, C1QBP, SPP1, VCAM1, NELL2, FLT3LG, ITGA4, TNFRSF10B, RUNX3,SERPING1, KLRC1, NLRC5, HPRT1, IKBKE, TNF, ADA, PDCD1, C1S, CCR2, TAB1,IL12RB1, IL2RA, PSEN2, LRP1, NOL7, SPN, TCF7, MFGE8, MST1R, IL10, LRRN3,PSMB9, PPIA, AGK, EDC3, CD80, PSMB8, CD276, BIRC5, TNFRSF9, TP53,ZC3H14, HLA-DRB3, ICOSLG, CD200, PIN1, EGR2, ITGAE, TRAF2, ABL1, ALCAM,IRF5, SF3A3, HRAS, NOD1, PDCD1LG2, IRF3, ANXA1, BASP1, BCL6, BLK, CAMP,CCL17, CCL22, CCR3, CEACAM1, CEACAM6, CFD, CR2, CSF2RB, CSF3R, CXCL1,CXCL6, CXCR1, CXCR2, FUT7, IFNL1, IL18RAP, IL19, IL1A, IL1RAP, IL1RN,IL8, ITGAX, LCN2, LILRB3, LYN, MAPK3, MEFV, MYD88, OSM, PBEF1, POLR2A,PRKCD, PTGS2, S100A12, S100A7, S100A8, SH2B2, SH2D1B, SPINK5, TFE3,TLR1, TNFRSF10C, TNFRSF11A, TNFRSF1A, TNFSF14, and TREM1.

In certain embodiments, provided herein are kits, systems, and/orcompositions comprising: a) a sample from a subject having a tissue ororgan graft, wherein the sample comprises urine from the subject; and b)reagent configured for detecting the level of at least one RNA markerselected from the group consisting of: C1QA, C1QB, C1R, CD3E, CTSL,CX3CL1, GSMA, HAVCR2, IF135, MSR1, SIGLEC1, and ENG.

In particular embodiments, provided herein are kits, systems, and/orcompositions comprising: a) a sample from a subject having a tissue ororgan graft, wherein the sample comprises urine from the subject; and b)reagent configured for detecting the level of at least one RNA markerselected from the group consisting of: CXCL9, CXCL10, CXCL11, LAG3,CD38, CD3D, IDOL CCL5, PRF1, KLRK1, TCRA, CTLA4, CD8A, STAT4, and CD27.In certain embodiments, the urine sample is from a subject having akidney tissue or organ graft.

DESCRIPTION OF THE FIGURES

FIG. 1 shows 49 genes that are upregulated by 5-fold or more in urinesediment of kidney transplant patients collected at the time of acuterejection (n=7) when compared to urine sediment collected fromtransplant recipients without rejection (n=14).

FIGS. 2A-H show 88 genes that were upregulated and 30 genes weredownregulated by at least two-fold in urine sediment of kidneytransplant patients collected at the time of acute rejection (n=17) whencompared to urine sediment collected from transplant recipients withoutrejection (n=29).

FIG. 3 shows the gene expression in the urine sediment at the time ofacute rejection in 12 subjects with biopsy-proven acute rejection and 10control subjects. Twenty-seven genes are upregulated by at least 5-foldin rejectors compared to controls and 21 genes are down-regulated by atleast 5-fold.

FIGS. 4A-4D show gene expression in graft tissue at the time of acuterejection in 9 subjects with acute rejection and 3 controls. One hundredfifty nine genes were upregulated but at least 5-fold in rejectinggrafts when compared with controls.

FIG. 5 shows genes upregulated by at least 5-fold at the time of acuterejection in the urine sediment and graft tissue of kidney graftrecipients.

FIG. 6 shows a volcano plot illustrating differential gene expression inurine sediment RNA from subjects with acute rejection compared withcontrol subjects.

FIG. 7 shows a gene signature of acute rejection in urine. Using theelastic net, a penalized regression method that applies both the lassoand ridge penalties, we determined the 10 genes that best distinguishurine RNA samples from subjects with and without acute rejection. A)Signature genes and weights in final algorithm; B) performance ofsignature in training set samples; C) ROC curves and mean AUC fromsplit-sample cross-validation.

FIG. 8 Panel A shows an exemplary 24 gene signature that distinguishesinjury from non-injury with an AUC of 0.825.

FIG. 8 Panel B shows a 55 gene signature that distinguishes injury dueto acute rejection from injury caused by BK virus nephropathy. In urineRNA samples that have been classified as “injured” using the 24 genesignature, the expression levels of these genes can identify the causeof the injury with an area under the curve of 0.962.

FIG. 9 shows a validation of the 10 gene acute rejection signature in aseparate set of samples. The left graph shows true positive rate vs.false positive rate; the center graph shows acute rejection vs. control;and the right graph shows ROC in test data.

DETAILED DESCRIPTION

Provided herein are compositions, systems, kits, and methods fordetecting rejection, or an elevated risk of rejection, of an organ ortissue graft (e.g., kidney graft) in a subject by detecting one, or apanel of, RNA markers in a urine sample from the subject. In certainembodiments, kidney graft injury is detected by measuring one or more ofthe RNA markers disclosed herein. In some embodiments, the one or moremarkers are employed to distinguish between acute graft rejection and BKvirus nephropathy.

In certain embodiments, the nanostring detection platform (NanoStringTechnologies) is employed to detect mRNA markers in a sample. Thenanostring platform technology uses molecular “barcodes” and singlemolecule imaging to detect and count hundreds of unique transcripts in asingle reaction. The resulting tests are able to identify geneticsequences at much smaller quantities and without the need for extensiveamplification of any genetic or proteomic material, which saves time andmoney. This process has been demonstrated to yield highly accurateresults.

Example 1

The NanoString nCounter platform was used to interrogate increases, aswell as decreases, in urine RNA targets to detect kidney graft injury.The NanoString nCounter technology allows multiplex detection of up to800 gene targets in a single reaction/assay; high sensitivity vs. theuse of microarray technology, high precision, and tolerance for RNAsamples that have been partially degraded such as those informalin-fixed/paraffin embedded tissue sections and urine. It is alsovery quick and the assays can be performed very cheaply.

Results

In initial analyses, we utilized urine samples collected from kidneytransplant patients enrolled in the CTOT-16 study; 14 from controlpatients and 7 from patients with acute rejection that were interrogatedby the NanoString platform. The gene expression analyses indicated that102 genes are upregulated by at least 2-fold during ongoing rejectionwith 49 of those upregulated by at least 5-fold (FIG. 1). These genesincluded those that had previously been identified in the urine by qPCR(Table 1), supporting the utility of the NanoString platform to quantifygene expression in urine RNA samples from kidney transplant patients.

TABLE 1 Fold- Gene Change CXCL10 25.6 CXCL9 13.9 Perforin 7.8Interferon-y 4.3 CD3E 4.3 Granzyme B 3.5 CXCR3 3.0 OX40 2.7 PD-1 2.6FOXP3 2.3 OX40L 2.3 Granulysin 2.3 TLR4 2.1To confirm these results in a larger set of subjects we obtainedadditional urine samples collected as part of the CTOT-10, CTOT-15 andCTOT-16 studies to bring our total sample set to 29 control and 17 acuterejection samples. The NanoString analyses indicated 88 genesupregulated by at least 2-fold during rejection vs. controls with 9upregulated by at least 5-fold and 30 genes down-regulated by at leasttwo-fold during acute rejection with 11 of those down-regulated by atleast 5-fold (FIGS. 2A-H).

Next, we performed a comparison of gene expression patterns in RNAisolated from urine vs. biopsy during acute kidney graft rejection usingurine RNA from 12 samples obtained during biopsy proven rejection and 10samples from control/non-rejecting grafts and RNA from kidney graftbiospies from 9 samples obtained during acute rejection and 3 obtainedfrom grafts without rejection. In the urine RNA samples 27 genes wereupregulated by 5-fold or greater at the time of clinically diagnosedrejection and 21 genes were down-regulated (FIG. 3). For the graftbiopsy RNA samples, 159 genes were upregulated by 5-fold or greater atthe time of rejection with no genes down-regulated (FIGS. 4A-4D). Of thegenes upregulated 5-fold or greater in urine and biopsy tissue duringongoing acute rejection (FIG. 5), 15 were common to both urine and grafttissue (Table 2) and 144 were unique to graft tissue and 12 were uniqueto urine (Table 3).

TABLE 2 Control Rejection Fold- samples samples change Gene (C) (AR)AR/C P value expression in urine CXCL9 5 269 52.8 4E−05 CXCL10 8 26134.1 6E−05 CXCL11 10 264 26.4 7E−05 LAG3 3 35 10.6 0.004 CD38 4 41 10.25E−07 CD3D 11 100 9.3 0.007 IDO1 45 408 9.1 0.004 CCL5 44 308 7.0 0.004PRF1 13 90 6.9 0.012 KLRK1 6 39 6.8 0.01 TCRA 27 185 6.8 0.04 CTLA4 7 405.9 0.002 CD8A 5 29 5.9 0.008 STAT4 8 46 5.7 0.001 CD27 7 37 5.3 0.03expression in tissue CXCL9 230 17,242 75.1 0.02 CXCL10 214 6,152 28.80.02 CXCL11 75 3,059 40.9 0.02 LAG3 2 208 109.0 6E−08 CD38 18 392 21.90.02 CD3D 50 1,078 21.5 0.02 IDO1 51 1,827 36.1 0.01 CCL5 60 904 15.00.03 PRF1 32 420 13.2 0.02 KLRK1 39 628 16.0 0.04 TCRA 225 4,026 17.90.006 CTLA4 4 296 78.1 0.02 CD8A 6 311 48.5 0.04 STAT4 31 203 6.6 0.01CD27 8 353 41.5 0.01

TABLE 3 Control Rejection Fold- samples samples change Gene (C) (AR)AR/C P value C1QA 9 77 8.1 0.00004 C1QB 71 363 5.1 0.005 C1R 3 21 6.00.001 CD3E 5 26 5.6 0.01 CTSL 109 665 6.1 0.02 CX3CL1 32 270 8.3 0.002GZMA 12 116 9.9 0.01 HAVCR2 27 144 5.3 0.004 IFI35 9 48 5.1 0.01 MSR1 25203 8.2 0.002 SIGLEC1 9 56 6.1 0.001 ENG 8 44 5.8 0.02Next, we took 29 of the urine RNA samples obtained from control/noclinical rejection recipients and 17 samples from recipients at the timeof ongoing rejection. The gene expression profiles were once againanalyzed by NanoString to obtain the volcano plot shown in FIG. 6indicating the mRNA from genes upregulated during rejection vs. controlson the right side of the plot and those down-regulated on the left sideof the plot.

Using the elastic net, a penalized regression method that applies boththe lasso and ridge penalties, we derived a set of 10 genes for theurine RNA signature of ongoing rejection (FIG. 7). The performance ofthis training set indicated an excellent separation from the control RNAsamples and the area under the curve (AUC) of receiver operatingcharacteristic (ROC) curve analysis was >84%.

It can be important to distinguish immune mediated graft rejection fromother causes of graft inflammation caused by infectious and otherinsults. We obtained urine RNA from 15 kidney transplant recipients withBK virus nephropathy and without clinical evidence of kidney graftrejection and the gene expression in these samples were compared to the17 RNA samples from patients with ongoing rejection by NanoString.Importantly, when compared with control samples, the most significantlyupregulated genes at the time of acute rejection were the same as thosemost upregulated during BK virus nephropathy (CXCL9, CXCL10, CXCL11,CX3CL1 and IDO1). Using the elastic net, we developed a set of 55 genesthat when measured together can distinguish transplant recipientsexperiencing acute rejection from transplant recipients with BK virusnephropathy (AUC >96%; 95% confidence interval 0.78-0.99 by ROC curveanalysis) (FIG. 8).

Finally, we obtained frozen urine sediment samples that had beencollected during the CTOT-08 trial. We chose 7 ARV visits withbiopsy-diagnosed T cell mediated kidney graft rejection and 15control/no clinical rejection samples and tested the gene expressionprofiles and the ability of the training set of 10 genes to detect thepresence vs. absence of rejection. The 10 gene test had a high TruePositive and low False Positive rate (FIG. 9), a high predictor score indistinguishing acute rejection from no rejection, and the area under thecurve (AUC) of receiver operating characteristic (ROC) curve analysiswas >98%. Overall these results validate the 10 gene test set for theability to detect clinically diagnosed T cell mediated rejection andsupport the use of this approach as a noninvasive diagnostic in kidneytransplant patients.

REFERENCES

-   -   1. Keslar et al., Am. J. Transplant, 13:1891-1897, 2013.    -   2. Hricik et al., Am. J. Transplant, 13:2634-2644, 2013.    -   3. Suthanthiran et al., N. Engl. J. Med., 369:20-31, 2013.    -   4. Hricik et al., J. Am. Soc. Nephrol., 26:3114-3122, 2015.    -   5. WO2015200873    -   6. WO2013173493.

All publications and patents mentioned in the specification and/orlisted below are herein incorporated by reference. Various modificationsand variations of the described method and system of the invention willbe apparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope described herein.

We claim:
 1. A method of detecting rejection, or an elevated risk ofrejection, or injury, of an organ or tissue graft in a subjectcomprising: detecting an upregulated level of at least one first RNAmarker in a sample from a subject and/or a downregulated level of atleast one second RNA marker in said sample from said subject, andthereby detecting acute rejection and/or elevated risk of rejection ofsaid organ or tissue graft, wherein said sample comprises urine fromsaid subject, wherein said subject has a tissue or organ graft, whereinsaid at least one first RNA marker that is detected as being upregulatedis selected from the group consisting of: CXCL11, CCL5, ETS1, LIF, TNF;and wherein said at least one second RNA marker that is detected asbeing downregulated is selected from the group consisting of: CCL17 andIL19.
 2. The method of claim 1, wherein said subject was taking a firstimmunosuppressant at the time said sample was obtained.
 3. The method ofclaim 2, further comprising the step of performing at least one of thefollowing: i) treating said subject with a second immunosuppressant thatis different from said first immunosuppressant; ii) generating and/ortransmitting a report that indicates said at least one first RNA makeris upregulated and/or said at least one second RNA marker isdownregulated in said sample, and/or that said subject is in need of animmunosuppressant different from said first immunosuppressant, and/or isin need of a having said tissue and/or organ graft removed or replaced;iii) generating and/or transmitting a report that indicates said atleast one first RNA maker is upregulated and/or said at least one secondRNA marker is downregulated in said sample, and/or that said subject hasacute rejection and/or elevated risk of rejection of said organ ortissue graft; and iv) characterizing said subject as having acuterejection and/or an elevated risk of rejection of said organ or tissuegraft, based on finding that said at least one first RNA maker isupregulated and/or said at least one second RNA marker is downregulatedin said sample.
 4. The method of claim 1, wherein said organ or tissuegraft comprises a kidney or kidney tissue graft.
 5. The method of claim1, wherein said subject at the time of collection of said sample wasbeing treated with, and/or wherein said sample comprises, animmunosuppressant protein that comprises an extracellular domain ofCTLA-4.
 6. The method of claim 5, wherein said immunosuppressant proteinfurther comprises an Fc region.
 7. The method of claim 5, wherein saidimmunosuppressant protein comprises Belatacept or Abatacept.
 8. Themethod of claim 1, wherein said detecting at least one of said firstand/or second RNA marker comprises detecting at least two of said firstand/or second RNA markers.
 9. The method of claim 1, further comprising:detecting an additional marker selected from upregulated TGFB1 and/orupregulated HLA.A.
 10. The method of claim 1, further comprising:detecting an additional marker selected from upregulated CXCL9 and/orupregulated CXCL10.
 11. The method of claim 1, wherein said detectingcomprise detecting all of the following: i) upregulated CXCL11, TGFB1,LIF, CCL5, TNF, HLA.A, and ETS1, and ii) downregulated IL19, CCL17, andIFIT1.
 12. A method of detecting rejection, or an elevated risk ofrejection, or injury of an organ or tissue graft in a subjectcomprising: detecting an upregulated level of at least one first RNAmarker in a sample from a subject and/or a downregulated level of atleast one second RNA marker in said sample from said subject, andthereby detecting acute rejection and/or elevated risk of rejection ofsaid organ or tissue graft, wherein said sample comprises urine fromsaid subject, wherein said subject has a tissue or organ graft, whereinsaid at least one first RNA marker that is detected as being upregulatedis selected from the group consisting of: TNFRSF11B, ETS1, CDK1, SIGIRR,CD274, CXCL11, ITGA6, and SF3A3, and wherein said at least one secondRNA marker that is detected as being downregulated, are selected fromthe group consisting of: TNFSF4, ATG16L1, MERTK, DHX16, CXCL14, IL19,CCL17, ATF1, CFD, IFIT1, CD163, ELK1, DPP4, and LAMP2.
 13. The method ofclaim 12, wherein said subject was taking a first immunosuppressant atthe time said sample was obtained.
 14. The method of claim 13, furthercomprising the step of performing at least one of the following: i)treating said subject with a second immunosuppressant that is differentfrom said first immunosuppressant; ii) generating and/or transmitting areport that indicates said at least one first RNA maker is upregulatedand/or said at least one second RNA marker is downregulated in saidsample, and/or that said subject is in need of an immunosuppressantdifferent from said first immunosuppressant, and/or is in need of ahaving said tissue and/or organ graft removed or replaced; iii)generating and/or transmitting a report that indicates said at least onefirst RNA maker is upregulated and/or said at least one second RNAmarker is downregulated in said sample, and/or that said subject hasacute rejection and/or elevated risk of rejection of said organ ortissue graft; and iv) characterizing said subject as having acuterejection and/or an elevated risk of rejection of said organ or tissuegraft, based on finding that said at least one first RNA maker isupregulated and/or said at least one second RNA marker is downregulatedin said sample.
 15. The method of claim 12, wherein said organ or tissuegraft comprises a kidney or kidney tissue graft.
 16. The method of claim12, wherein said subject has been treated with an anti-CD52 monoclonalantibody or antigen binding fragment thereof.
 17. The method of claim12, wherein said subject at the time of collection of said sample wasbeing treated with, and/or wherein said sample comprises, animmunosuppressant comprising tacrolimus (aka FK506).
 18. The method ofclaim 12, wherein said detecting at least one of said first and/orsecond RNA marker comprises detecting at least two of said first and/orsecond RNA markers.
 19. The method of claim 12, further comprising:detecting an additional marker selected from upregulated CXCL9 and/orupregulated CXCL10.
 20. A method of detecting rejection or BK virusinfection, or an elevated risk of rejection, or injury, of an organ ortissue graft in a subject comprising: detecting an upregulated level ofat least one first RNA marker in a sample from a subject and/or adownregulated level of at least one second RNA marker in said samplefrom said subject, and thereby detecting acute rejection and/or elevatedrisk of rejection of said organ or tissue graft, wherein said samplecomprises urine from said subject, wherein said subject has a tissue ororgan graft, wherein said at least one first RNA marker that is detectedas being upregulated and is selected from the group consisting of:SIGLEC1, CD163, PSEN1, DPP4, CEACAM8, PYCARD, MTMR14, ICOSLG, NOL7, C2,HLA.DRB3, CD68, ST6GAL1, SLAMF1, MERTK, PRAME, ELK1, STAT4, CD99, CD86,LAMP2, ITGB1, IL21R, IL6ST, TNF, GPATCH3, GPI, ATF1, PIN1, and YTHDF2,and wherein said at least one second RNA marker that is detected asbeing downregulated, is selected from the group consisting of: HMGB1,MAPK9, SPA17, TNFRSF11B, GTF3C1, BST2, PRPF38A, MAF, BATF, EGFR, CD274,CCL17, SERPINA3, GBP1, and PBEF1.
 21. The method of claim 20, wherein BKvirus infection is detected.
 22. The method of claim 20, whereinrejection is detected.
 23. The method of claim 20, wherein said organ ortissue graft comprises a kidney or kidney tissue graft.
 24. The methodof claim 20, further comprising: detecting an additional marker selectedfrom the group consisting of: C1QBP, CD6, CXCL6, LIF, AXL, CREBBP,PSMB7, NFATC1, TGFB1, and FCER1G.